Extending Linker Sequences between Antigen-Recognition Modules Provides More Effective Production of Bispecific Nanoantibodies in the Periplasma of E. coli

Technology of production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. Production of nAb in periplasm of E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and selection of the most promising nAb variants. The situation is more complicated with production of bi- and multivalent derivatives of the initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between the antigen-recognition modules in the cloned expression constructs were developed and applied in order to increase efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of functionality of all the protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of original monomeric nAbs. The use of sufficiently long linkers could presumably be useful for increasing efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.